Interfering rna delivery system and uses thereof

ABSTRACT

The invention provides a delivery system comprising a cell penetrating peptide, a polyarginine peptide, and an interfering RNA molecule. The system can be used for delivering interfering RNA molecules into a cell in vivo or in vitro. Therapeutic uses for the delivery system are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a divisional of U.S. patent application Ser.No. 12/967,182 filed Dec. 14, 2010, which claims benefit to U.S. patentapplication Ser. No. 12/338,252 filed Dec. 18, 2008, which claimsbenefit to U.S. Provisional Patent Application Ser. No. 61/014,472 filedDec. 18, 2007, the entire contents of which are incorporated herein byreference.

FIELD OF THE INVENTION

The invention relates to a delivery system for delivering interferingRNA molecules into a cell and methods for using the delivery system. Thedelivery system comprises a TAT peptide, or other cell-penetratingpeptide, and a polyarginine peptide, is such as a 9× Arg peptide. Thedelivery system can be administered to a cell in the presence of an HA2peptide, which can be incorporated into the system or administeredconcurrently with the system.

BACKGROUND OF THE INVENTION

RNA interference (RNAi) is a process by which double-stranded RNA(dsRNA) is used to silence gene expression. RNAi is induced by short(i.e. <30 nucleotide) double stranded RNA (“dsRNA”) molecules which arepresent in the cell (Fire et al., 1998, Nature 391:806-811). These shortdsRNA molecules called “short interfering RNA” or “siRNA,” cause thedestruction of messenger RNAs (“mRNAs”) which share sequence homologywith the siRNA (Elbashir et al., 2001, Genes Dev, 15:188-200). It isbelieved that one strand of the siRNA is incorporated into aribonucleoprotein complex known as the RNA-induced silencing complex(RISC). RISC uses this siRNA strand to identify mRNA molecules that areat least partially complementary to the incorporated siRNA strand, andthen cleaves these target mRNAs or inhibits their translation. The siRNAis apparently recycled much like a multiple-turnover enzyme, with 1siRNA molecule capable of inducing cleavage of approximately 1000 mRNAmolecules. siRNA-mediated RNAi degradation of an mRNA is therefore moreeffective than currently available technologies for inhibitingexpression of a target gene.

RNAi provides a very exciting approach to treating and/or preventingdiseases. Some major benefits of RNAi compared with various traditionaltherapeutic approaches include: the ability of RNAi to target a veryparticular gene involved in the disease process with high specificity,thereby reducing or eliminating off target effects; RNAi is a normalcellular process leading to a highly specific RNA degradation; and RNAidoes not trigger a host immune response as in many antibody basedtherapies.

Several interfering RNA delivery methods are being tested/developed forin vivo use. For example, siRNAs can be delivered “naked” in salinesolution; complexed with polycations, cationic lipids/lipid transfectionreagents, or cationic peptides; as components of defined molecularconjugates (e.g., cholesterol-modified siRNA, TAT-DRBD/siRNA complexes);as components of liposomes; and as components of nanoparticles. Theseapproaches have shown varying degrees of success. Thus, there remains aneed for new and improved methods for delivering siRNA molecules in vivoto achieve and enhance the therapeutic potential of RNAi.

Several cell-penetrating peptides (CPPs) or membrane permeant peptides(MPPs) have been described (Jarver and Langel, 2004, Drug Discov Today9:395-402) as conjugates to deliver peptides into cells. The proteintransduction domain (PTD) of the HIV-1 TAT protein is a CPP that appearsto be particularly effective. The TAT peptide has been used to deliverbiologically active cargo to cells in vitro and in vivo (Wadia andDowdy, 2003, Curr Protein Pept Sci. 4:97-104; Bullok et al., 2006, MolImaging 5:1-15).

Several groups have explored the use of CPPs to deliver interfering RNAmolecules (See Meade and Dowdy, 2007, Adv Drug Deliv Rev. 59:134-140).The main challenge to this approach involves linking the interfering RNAto the CPPs while maintaining the ability of the complex to interactwith and enter the intracellular environment. In particular, thenegative charge of the interfering RNA neutralizes the positivelycharged CPPs, which renders such complexes incapable of cellulardelivery. Thus, there is a need to identify ways to link CPPs tointerfering RNA molecules without hindering the ability of the CPP tofacilitate intracellular delivery of the interfering RNA.

SUMMARY OF THE INVENTION

The invention provides an interfering RNA delivery system comprising aninterfering RNA molecule linked to a CPP-Arg peptide, such as a TAT-9×Arg peptide. The invention also provides a method for delivering aninterfering RNA molecule into a cell, in vitro or in vivo, comprising:(a) attaching an interfering RNA molecule to a CPP-Arg peptide, therebyforming an interfering RNA delivery system; and administering the systemto the cell under conditions suitable for the system to enter the cell.In certain aspects, the delivery system is administered to a cell in thepresence of an HA2 peptide, which aids release of the system from theendosome. The HA2 peptide can be incorporated into the delivery systemor administered before, after, or with administration of the deliverysystem.

In one aspect, an interfering RNA molecule in a delivery system of theinvention can attenuate expression of a target mRNA in a target cell.Thus, the invention provides methods for attenuating expression of atarget mRNA in a cell comprising administering a delivery system of theinvention to the cell.

The invention further provides pharmaceutical compositions comprising aninterfering RNA delivery system of the invention. The pharmaceuticalcompositions can be used in therapeutic applications to treat variousdisorders or diseases in which inhibition of a target gene is desired.

In addition, the invention provides methods of treating or preventing anocular disorder in a patient, comprising administering to the patient aninterfering RNA delivery system as described herein to the patient,wherein the interfering RNA molecule can attenuate expression of a geneassociated with the ocular disorder. In certain aspects, the oculardisorder is associated with ocular angiogenesis, dry eye, ocularinflammatory conditions, ocular hypertension, or glaucoma. In otheraspects, the conjugate is administered by intraocular injection, oculartopical application, subconjunctival injection, intravitreal injection,anterior or posterior juxtascleral injection, intravenous injection,oral administration, intramuscular injection, intraperitoneal injection,transdermal application, intranasal application, or transmucosalapplication.

Specific preferred embodiments of the invention will become evident fromthe following more detailed description of certain preferred embodimentsand the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts results of FACS analysis of non-treated GTM-3 cells.

FIG. 1B depicts results of FACS analysis of GTM-3 cells treated with ansiRNA labeled with Dy547 (Dy547-siRNA).

FIG. 1C depicts results of FACS analysis of GTM-3 cells treated with 9×Arg/Dy547-siRNA.

FIG. 1D depicts results of FACS analysis of GTM-3 cells treated withPen7-9× Arg/Dy547-siRNA.

DETAILED DESCRIPTION OF THE INVENTION

The particulars shown herein are by way of example and for purposes ofillustrative discussion of the preferred embodiments of the presentinvention only and are presented in the cause of providing what isbelieved to be the most useful and readily understood description of theprinciples and conceptual aspects of various embodiments of theinvention. In this regard, no attempt is made to show structural detailsof the invention in more detail than is necessary for the fundamentalunderstanding of the invention, the description taken with the drawingsand/or examples making apparent to those skilled in the art how theseveral forms of the invention may be embodied in practice.

The following definitions and explanations are meant and intended to becontrolling in any future construction unless clearly and unambiguouslymodified in the following examples or when application of the meaningrenders any construction meaningless or essentially meaningless. Incases where the construction of the term would render it meaningless oressentially meaningless, the definition should be taken from Webster'sDictionary, 3^(rd) Edition or a dictionary known to those of skill inthe art, such as the Oxford Dictionary of Biochemistry and MolecularBiology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).

As used herein, all percentages are percentages by weight, unless statedotherwise.

As used herein and unless otherwise indicated, the terms “a” and “an”are taken to mean “one”, “at least one” or “one or more”. Unlessotherwise required by context, singular terms used herein shall includepluralities and plural terms shall include the singular.

In certain embodiments, the invention provides an interfering RNAdelivery system comprising an interfering RNA molecule linked to a cellpenetrating peptide (CPP) via an Arg peptide.

As used herein, the phrase “interfering RNA delivery system” refers to asystem that comprises a CPP, an Arg peptide, and an interfering RNAmolecule, which is capable of delivering the interfering RNA moleculeinto a cell. In certain embodiments, the interfering RNA delivery systemcan be administered to a subject in need thereof.

The term “CPP-Arg peptide” as used herein refers to a peptide comprisinga CPP linked to an Arg peptide. For example, a “TAT-Arg peptide” means apeptide comprising a TAT peptide linked to an Arg peptide.

An “Arg peptide” or “polyarginine” is a peptide consisting entirely ofarginines. Preferably, the Arg peptide comprises 7, 8, 9, 10, or 11arginines. In certain embodiments, the Arg peptide will be linked to theC- or N-terminus of TAT via a glycine spacer of 1, 2, 3, or 4 glycines.Preferably, the glycine spacer is 2 or 3 glycines.

In one embodiment, the Arg peptide is a 9× Arg peptide. The term “9× Argpeptide” as used herein means a peptide comprising 9 arginine residues(RRRRRRRRR; SEQ ID NO: 1). In one embodiment, the 9× Arg peptidecomprises or consists of D-isomers. Negatively charged interfering RNAmolecules can bind to the positively charged 9× Arg peptide as describedin Kumar et al., who recently demonstrated that a 9× Arg peptide couldbe used to link interfering RNA molecules to the C-terminal end of arabies virus glycoprotein (RVG) targeting peptide for delivery acrossthe blood-brain barrier (Kumar et al., Jun. 17, 2007, Nature, epub aheadof print).

The terms “cell penetrating peptide” and “CPP” as used herein refer topeptides, typically of about 9 to about 30 amino acid residues, capableof being internalized by a mammalian cell. For example, a CPP can be aprotein transduction domain or a fragment thereof, as discussed below.In most instances, a CPP enters the intracellular environment byendocytosis. In other instances, a CPP may remain in the cell membrane,while facilitating its cargo to enter the cell. Examples of useful CPPsinclude, but are not limited to, the TAT peptide, and the proteintransduction domains of Penetratin (pAntp), Transportan, MPG,MPGdeltaNLS, and pHLIP. Cell penetrating fragments of CPPs can also beused in a delivery system and/or method of the invention. As usedherein, the term CPP includes cell penetrating fragments of proteintransduction domains.

In certain embodiments, a CPP can comprise or consist of D-amino acidsand/or L-amino acids. For example, a CPP can consist entirely of D-aminoacids or entirely of L-amino acids; or a CPP can comprise a mixture ofD- and L-amino acids.

In certain embodiments, the amino acid sequence of a CPP can be in theforward direction (i.e. a native peptide) or in the reverse direction.As used herein, reference to a CPP includes both the native and reversesequences. In one embodiment, the reverse sequence can be aretro-inverso peptide (i.e. the amino acid sequence is the reverse ofthe native sequence, and consists of D-amino acids). For example, theterm “TAT peptide” as used herein includes a retro-inverso TAT peptidecomprising a reverse sequence of the protein transduction domain (PTD)of the HIV-1 TAT protein, as illustrated below. The native sequence PTDof TAT is:

SEQ ID NO: 2 YGRKKRRQRRR; (Vives et al., 1997, J. Biol. Chem 272:16010).A retro-inverso TAT peptide (i.e., the reverse sequence constructed ofD-amino acids) is:

SEQ ID NO: 3 R^(†)R^(†)R^(†)Q^(†)R^(†)R^(†)K^(†)K^(†)R^(†)G^(†);.

D-isomers are denoted by a superscripted dagger (^(†)) to the right ofthe one-letter code symbol; thus, D^(†) represents D-aspartic acid andL^(†) represents D-leucine.

The PTD of Penetratin (pAntp) is:

SEQ ID NO: 4 RQIKIWFQNRRMKWKK; (Muratovska and Eccles, 2004,FEBS Letters 558:63-68).

The PTD of Transportan is:

SEQ ID NO:5 LIKKALAALAKLNIKLLYGASNLTWG; (Muratovska and Eccles, 2004,FEBS Letters 558:63-68).

The PTD of MPG is:

SEQ ID NO: 6 GALFLGFLGAAGSTMGAWSQPKKKRKV; (Simeoni et al., 2003,Nucl. Acids Res. 31:2717-2724).

The PTD of MPGdeltaNLS is:

SEQ ID NO: 7 GALFLGFLGAAGSTMGAWSQPKSKRKV; (Simeoni et al., 2003,Nucl. Acids Res. 31:2717-2724).

The PTD of pHLIP is:

SEQ ID NO: 8 ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG;(Andreev et al., 2007, Proc. Natl. Acad. Sci. USA 104:7893-7899).

In certain embodiments, an interfering RNA delivery system isadministered to a subject or a cell in the presence of a TAT-HA2peptide, a ligand-HA2 peptide, or a retro-inverso TAT-HA2 peptide, whichhas been shown to enhance release of TAT-peptide/protein from theendosome (Wadia et al., 2004, Nat. Med. 10:310). The term “HA2 peptide”means a peptide comprising the N-terminal 20 amino acids of influenzavirus hemagglutinin protein. The native HA2 peptide is:

SEQ ID NO: 9 GLFGAIAGFIENGWEGMIDG;.Preferably, the native HA2 peptide comprises L-isomers, but may beconstructed to comprise or consist of D-amino acids.

The retro-inverso HA2 peptide is:

SEQ ID NO: 10GD^(†)I^(†)M^(†)GE^(†)W^(†)GN^(†)E^(†)I^(†)F^(†)GA^(†)I^(†)A^(†)GF^(†)L^(†)G;.

The retro-inverso HA2 peptide may comprise or consist of D-amino acids.

The presence of HA2 aids release of the interfering RNA delivery systemfrom the endosome into the cytosol, so that the interfering RNAmolecular can attenuate expression of a target mRNA in the subject orcell. In certain other embodiments, an HA2 peptide is inserted betweenthe CPP peptide and a 9× Arg, wherein the HA2 peptide is linked to the9× Arg via a glycine spacer. For example, where TAT is the CPP, thesequence may be as follows:

SEQ ID NO: 11R^(†)R^(†)R^(†)Q^(†)R^(†)R^(†)K^(†)K^(†)R^(†)GGGD^(†)I^(†)M^(†)GE^(†)W^(†)GN^(†)E^(†)I^(†)F^(†)GA^(†)I^(†)A^(†)GF^(†)L^(†)GGGR^(†)R^(†)R^(†)R^(†)R^(†)R^(†)R^(†)R^(†)R^(†);.

In certain embodiments, the CPP-Arg peptide is produced as a singlepeptide before being conjugated to the interfering RNA molecule. Inother embodiments, the CPP-Arg peptide can be produced by combining theCPP (or CPP-HA2) peptide and the Arg peptide under conditions in whichthe two peptides will connect to each other. Such methods for linkingtwo peptides are well known in the art. In yet other embodiments, theArg peptide can be premixed with the interfering RNA molecule and thenlinked to the CPP (or CPP-HA2) peptide to favor binding of theinterfering RNA to the Arg end of the peptide. Thus, linkage of theinterfering RNA molecule can be accomplished before or after linkage ofCPP (or CPP-HA2) with Arg.

In certain embodiments, an interfering RNA delivery system as describedherein can be used in a method of delivering an interfering RNA moleculeinto a cell. The cell can be an isolated cell (e.g. in cell culture) orassociated with a subject in which inhibiting expression of a targetgene is desired. The cell may also be used in a ex vivo therapeuticmethod, in which the cell is taken from a subject and reintroduced intothe same or a different subject after the interfering RNA deliverysystem has been introduced into the cell.

As used herein, the term “subject” or “patient” refers to human andnon-human animals. The term “non-human animals” refers to vertebratesand non-vertebrates, including but, not limited to, primates, rabbits,pigs, horses, dogs, cats, sheep, and cows. In one embodiment, a patienthas an ocular disorder or is at risk of having an ocular disorder.Ocular structures associated with such disorders may include the eye,retina, choroid, lens, cornea, trabecular meshwork, iris, optic nerve,optic nerve head, sclera, anterior or posterior segment, or ciliarybody, for example. In certain embodiments, a patient has an oculardisorder associated with trabecular meshwork (TM) cells, ciliaryepithelium cells, or another cell type of the eye.

The term “ocular disorder” as used herein includes conditions associatedwith ocular angiogenesis, dry eye, inflammatory conditions, ocularhypertension and ocular diseases associated with elevated intraocularpressure (TOP), such as glaucoma.

The term “ocular angiogenesis,” as used herein, includes ocularpre-angiogenic conditions and ocular angiogenic conditions, and includesocular angiogenesis, ocular neovascularization, retinal edema, diabeticretinopathy, sequela associated with retinal ischemia, posterior segmentneovascularization (PSNV), and neovascular glaucoma, for example. Theinterfering RNAs used in a method of the invention are useful fortreating patients with ocular angiogenesis, ocular neovasularization,retinal edema, diabetic retinopathy, sequela associated with retinalischemia, posterior segment neovascularization (PSNV), and neovascularglaucoma, or patients at risk of developing such conditions, forexample. The term “ocular neovascularization” includes age-relatedmacular degeneration, cataract, acute ischemic optic neuropathy (AION),commotio retinae, retinal detachment, retinal tears or holes, iatrogenicretinopathy and other ischemic retinopathies or optic neuropathies,myopia, retinitis pigmentosa, and/or the like.

The term “inflammatory condition,” as used herein, includes conditionssuch as ocular inflammation and allergic conjunctivitis.

The interfering RNA delivery system of the invention is useful forattenuating expression of particular genes in a patient (i.e. subject)using RNA interference.

RNA interference (RNAi) is a process by which double-stranded RNA(dsRNA) is used to silence gene expression. While not wanting to bebound by theory, RNAi begins with the cleavage of longer dsRNAs intosmall interfering RNAs (siRNAs) by an RNaseIII-like enzyme, dicer.siRNAs are dsRNAs that are usually about 19 to 28 nucleotides, or 20 to25 nucleotides, or 21 to 22 nucleotides in length and often contain2-nucleotide 3′ overhangs, and 5′ phosphate and 3′ hydroxyl termini. Onestrand of the siRNA is incorporated into a ribonucleoprotein complexknown as the RNA-induced silencing complex (RISC). RISC uses this siRNAstrand to identify mRNA molecules that are at least partiallycomplementary to the incorporated siRNA strand, and then cleaves thesetarget mRNAs or inhibits their translation. Therefore, the siRNA strandthat is incorporated into RISC is known as the guide strand or theantisense strand. The other siRNA strand, known as the passenger strandor the sense strand, is eliminated from the siRNA and is at leastpartially homologous to the target mRNA. Those of skill in the art willrecognize that, in principle, either strand of an siRNA can beincorporated into RISC and function as a guide strand. However, siRNAdesign (e.g., decreased siRNA duplex stability at the 5′ end of thedesired guide strand) can favor incorporation of the desired guidestrand into RISC.

The antisense strand of an siRNA is the active guiding agent of thesiRNA in that the antisense strand is incorporated into RISC, thusallowing RISC to identify target mRNAs with at least partialcomplementarity to the antisense siRNA strand for cleavage ortranslational repression. RISC-mediated cleavage of mRNAs having asequence at least partially complementary to the guide strand leads to adecrease in the steady state level of that mRNA and of the correspondingprotein encoded by this mRNA. Alternatively, RISC can also decreaseexpression of the corresponding protein via translational repressionwithout cleavage of the target mRNA.

Interfering RNAs appear to act in a catalytic manner for cleavage oftarget mRNA, i.e., interfering RNA is able to effect inhibition oftarget mRNA in substoichiometric amounts. As compared to antisensetherapies, significantly less interfering RNA is required to provide atherapeutic effect under such cleavage conditions.

In certain embodiments, the invention provides methods of deliveringinterfering RNA to inhibit the expression of a target mRNA thusdecreasing target mRNA levels in patients with target mRNA-relateddisorders.

The phrase “attenuating expression” with reference to a gene or an mRNAas used herein means administering or expressing an amount ofinterfering RNA (e.g., an siRNA) to reduce translation of a target mRNAinto protein, either through mRNA cleavage or through direct inhibitionof translation. The terms “inhibit,” “silencing,” and “attenuating” asused herein refer to a measurable reduction in expression of a targetmRNA or the corresponding protein as compared with the expression of thetarget mRNA or the corresponding protein in the absence of aninterfering RNA of the invention. The reduction in expression of thetarget mRNA or the corresponding protein is commonly referred to as“knock-down” and is reported relative to levels present followingadministration or expression of a non-targeting control RNA (e.g., anon-targeting control siRNA). Knock-down of expression of an amountincluding and between 50% and 100% is contemplated by embodimentsherein. However, it is not necessary that such knock-down levels beachieved for purposes of the present invention.

Knock-down is commonly assessed by measuring the mRNA levels usingquantitative polymerase chain reaction (qPCR) amplification or bymeasuring protein levels by western blot or enzyme-linked immunosorbentassay (ELISA). Analyzing the protein level provides an assessment ofboth mRNA cleavage as well as translation inhibition. Further techniquesfor measuring knock-down include RNA solution hybridization, nucleaseprotection, northern hybridization, gene expression monitoring with amicroarray, antibody binding, radioimmunoassay, and fluorescenceactivated cell analysis.

Attenuating expression of a target gene by an interfering RNA moleculeof the invention can be inferred in a human or other mammal by observingan improvement in symptoms of the disorder.

In one embodiment, a single interfering RNA is delivered to decreasetarget mRNA levels. In other embodiments, two or more interfering RNAstargeting the mRNA are administered to decrease target mRNA levels. Theinterfering RNAs may be delivered through linkage to the same CPP-Argpeptide or through linkage to separate CPP-Arg peptide(s) (e.g. eachinterfering RNA can be pre-mixed and added to CPP-Arg or eachinterfering RNA can be independently mixed with CPP-Arg, followed bycombining the individual interfering RNA/CPP-Arg complexes).

As used herein, the terms “interfering RNA” and “interfering RNAmolecule” refer to all RNA or RNA-like molecules that can interact withRISC and participate in RISC-mediated changes in gene expression.Examples of other interfering RNA molecules that can interact with RISCinclude short hairpin RNAs (shRNAs), single-stranded siRNAs, microRNAs(miRNAs), picoRNAs (piRNAs), and dicer-substrate 27-mer duplexes.Examples of “RNA-like” molecules that can interact with RISC includesiRNA, single-stranded siRNA, miRNA, piRNA, and shRNA molecules thatcontain one or more chemically modified nucleotides, one or morenon-nucleotides, one or more deoxyribonucleotides, and/or one or morenon-phosphodiester linkages. Thus, siRNAs, single-stranded siRNAs,shRNAs, miRNAs, piRNA, and dicer-substrate 27-mer duplexes are subsetsof “interfering RNAs” or “interfering RNA molecules.”

The term “siRNA” as used herein refers to a double-stranded interferingRNA unless otherwise noted. Typically, an siRNA used in a method of theinvention is a double-stranded nucleic acid molecule comprising twonucleotide strands, each strand having about 19 to about 28 nucleotides(i.e. about 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides).Typically, an interfering RNA used in a method of the invention has alength of about 19 to 49 nucleotides. The phrase “length of 19 to 49nucleotides” when referring to a double-stranded interfering RNA meansthat the antisense and sense strands independently have a length ofabout 19 to about 49 nucleotides, including interfering RNA moleculeswhere the sense and antisense strands are connected by a linkermolecule.

The interfering RNA used in a delivery system and method of theinvention can be unmodified or can be chemically stabilized to preventdegradation in the lysosome or other compartments in the endocyticpathway.

Single-stranded interfering RNA has been found to effect mRNA silencing,albeit less efficiently than double-stranded RNA. Therefore, embodimentsof the present invention also provide for administration of asingle-stranded interfering RNA. The single-stranded interfering RNA hasa length of about 19 to about 49 nucleotides as for the double-strandedinterfering RNA cited above. The single-stranded interfering RNA has a5′ phosphate or is phosphorylated in situ or in vivo at the 5′ position.The term “5′ phosphorylated” is used to describe, for example,polynucleotides or oligonucleotides having a phosphate group attachedvia ester linkage to the C5 hydroxyl of the sugar (e.g., ribose,deoxyribose, or an analog of same) at the 5′ end of the polynucleotideor oligonucleotide.

Single-stranded interfering RNAs can be synthesized chemically or by invitro transcription or expressed endogenously from vectors or expressioncassettes as described herein in reference to double-strandedinterfering RNAs. 5′ Phosphate groups may be added via a kinase, or a 5′phosphate may be the result of nuclease cleavage of an RNA. A hairpininterfering RNA is a single molecule (e.g., a single oligonucleotidechain) that comprises both the sense and antisense strands of aninterfering RNA in a stem-loop or hairpin structure (e.g., a shRNA). Forexample, shRNAs can be expressed from DNA vectors in which the DNAoligonucleotides encoding a sense interfering RNA strand are linked tothe DNA oligonucleotides encoding the reverse complementary antisenseinterfering RNA strand by a short spacer. If needed for the chosenexpression vector, 3′ terminal T's and nucleotides forming restrictionsites may be added. The resulting RNA transcript folds back onto itselfto form a stem-loop structure.

Interfering RNAs may differ from naturally-occurring RNA by theaddition, deletion, substitution or modification of one or morenucleotides. Non-nucleotide material may be bound to the interferingRNA, either at the 5′ end, the 3′ end, or internally. Such modificationsare commonly designed to increase the nuclease resistance of theinterfering RNAs, to improve cellular uptake, to enhance cellulartargeting, to assist in tracing the interfering RNA, to further improvestability, to reduce off-target effects, or to reduce the potential foractivation of the interferon pathway. For example, interfering RNAs maycomprise a purine nucleotide at the ends of overhangs. Conjugation ofcholesterol to the 3′ end of the sense strand of an siRNA molecule bymeans of a pyrrolidine linker, for example, also provides stability toan siRNA.

Further modifications include a biotin molecule, a peptidomimetic, afluorescent dye, or a dendrimer, for example.

Nucleotides may be modified on their base portion, on their sugarportion, or on the phosphate portion of the molecule and function inembodiments of the present invention. Modifications includesubstitutions with alkyl, alkoxy, amino, deaza, halo, hydroxyl, thiolgroups, or a combination thereof, for example. Nucleotides may besubstituted with analogs with greater stability such as replacing aribonucleotide with a deoxyribonucleotide, or having sugar modificationssuch as 2′ OH groups replaced by 2′ amino groups, 2′ O-methyl groups, 2′methoxyethyl groups, or a 2′-O, 4′-C methylene bridge, for example.Examples of a purine or pyrimidine analog of nucleotides include axanthine, a hypoxanthine, an azapurine, a methylthioadenine,7-deaza-adenosine and O- and N-modified nucleotides. The phosphate groupof the nucleotide may be modified by substituting one or more of theoxygens of the phosphate group with nitrogen or with sulfur(phosphorothioates). Modifications are useful, for example, to enhancefunction, to improve stability or permeability, to reduce off-targeteffects, or to direct localization or targeting.

In certain embodiments, an interfering molecule of the inventioncomprises at least one of the modifications as described above.

The phrases “target sequence” and “target mRNA” as used herein refer tothe mRNA or the portion of the mRNA sequence that can be recognized byan interfering RNA used in a method of the invention, whereby theinterfering RNA can silence gene expression as discussed herein.Techniques for selecting target sequences for siRNAs are provided, forexample, by Tuschl, T. et al., “The siRNA User Guide,” revised May 6,2004, available on the Rockefeller University web site; by TechnicalBulletin #506, “siRNA Design Guidelines,” Ambion Inc. at Ambion's website; and by other web-based design tools at, for example, theInvitrogen, Dharmacon, Integrated DNA Technologies, or Genscript websites. Initial search parameters can include G/C contents between 35%and 55% and siRNA lengths between 19 and 27 nucleotides. The targetsequence may be located in the coding region or in the 5′ or 3′untranslated regions of the mRNA. The target sequences can be used toderive interfering RNA molecules, such as those described herein.

Interfering RNA target sequences (e.g., siRNA target sequences) within atarget mRNA sequence are selected using available design tools asdiscussed above. Interfering RNAs corresponding to a target sequence arethen tested in vitro by transfection of cells expressing the target mRNAfollowed by assessment of knockdown as described herein. The interferingRNAs can be further evaluated in vivo using animal models as describedherein.

The ability of interfering RNA to knock-down the levels of endogenoustarget gene expression in, for example, HeLa cells can be evaluated invitro as follows. HeLa cells are plated 24 h prior to transfection instandard growth medium (e.g., DMEM supplemented with 10% fetal bovineserum). Transfection is performed using, for example, Dharmafect 1(Dharmacon, Lafayette, Colo.) according to the manufacturer'sinstructions at interfering RNA concentrations ranging from 0.1 nM-100nM. SiCONTROL™ Non-Targeting siRNA #1 and siCONTROL™ Cyclophilin B siRNA(Dharmacon) are used as negative and positive controls, respectively.Target mRNA levels and cyclophilin B mRNA (PPIB, NM_(—)000942) levelsare assessed by qPCR 24 h post-transfection using, for example, aTAQMAN® Gene Expression Assay that preferably overlaps the target site(Applied Biosystems, Foster City, Calif.). The positive control siRNAgives essentially complete knockdown of cyclophilin B mRNA whentransfection efficiency is 100%. Therefore, target mRNA knockdown iscorrected for transfection efficiency by reference to the cyclophilin BmRNA level in cells transfected with the cyclophilin B siRNA. Targetprotein levels may be assessed approximately 72 h post-transfection(actual time dependent on protein turnover rate) by western blot, forexample. Standard techniques for RNA and/or protein isolation fromcultured cells are well-known to those skilled in the art. To reduce thechance of non-specific, off-target effects, the lowest possibleconcentration of interfering RNA is used that produces the desired levelof knock-down in target gene expression. Human corneal epithelial cellsor other human ocular cell lines may also be use for an evaluation ofthe ability of interfering RNA to knock-down levels of an endogenoustarget gene.

In certain embodiments, an interfering RNA delivery system comprises aninterfering RNA molecule that targets a gene associated with an oculardisorder. Examples of mRNA target genes for which interfering RNAs ofthe present invention are designed to target include genes associatedwith the disorders that affect the retina, genes associated withglaucoma, and genes associated with ocular inflammation.

Examples of mRNA target genes associated with the retinal disordersinclude TEK tyrosine kinase, endothelial (TEK); complement factor B(CFB); hypoxia-inducible factor 1, α subunit (HIF1A); HtrA serinepeptidase 1 (HTRA1); platelet-derived growth factor receptor β (PDGFRB);chemokine, CXC motif, receptor 4 (CXCR4); insulin-like growth factor Ireceptor (IGF1R); angiopoietin 2 (ANGPT2); v-fos FBJ murine osteosarcomaviral oncogene homolog (FOS); cathepsin L1, transcript variant 1(CTSL1); cathepsin L1, transcript variant 2 (CTSL2); intracellularadhesion molecule 1 (ICAM1); insulin-like growth factor I (IGF1);integrin α5 (ITGA5); integrin β1 (ITGB1); nuclear factor kappa-B,subunit 1 (NFKB 1); nuclear factor kappa-B, subunit 2 (NFKB2);chemokine, CXC motif, ligand 12 (CXCL12); tumor necrosis factor receptor1 (TNFR1); vascular endothelial growth factor (VEGF); vascularendothelial growth factor receptor 1 (VEGFR1); tumor necrosisfactor-alpha-converting enzyme (TACE); and kinase insert domain receptor(KDR).

Examples of target genes associated with glaucoma include carbonicanhydrase II (CA2); carbonic anhydrase IV (CA4); carbonic anhydrase XII(CA12); β1 andrenergic receptor (ADBR1); β2 andrenergic receptor(ADBR2); acetylcholinesterase (ACHE); Na+/K+-ATPase; solute carrierfamily 12 (sodium/potassium/chloride transporters), member 1 (SLC12A1);solute carrier family 12 (sodium/potassium/chloride transporters),member 2 (SLC12A2); connective tissue growth factor (CTGF); serumamyloid A (SAA); secreted frizzled-related protein 1 (sFRP1); gremlin(GREM1); lysyl oxidase (LOX); c-Maf; rho-associatedcoiled-coil-containing protein kinase 1 (ROCK1); rho-associatedcoiled-coil-containing protein kinase 2 (ROCK2); plasminogen activatorinhibitor 1 (PAT-1); endothelial differentiation, sphingolipidG-protein-coupled receptor, 3 (Edg3 R); myocilin (MYOC); NADPH oxidase 4(NOX4); Protein Kinase Cδ (PKCδ); Aquaporin 1 (AQP1); Aquaporin 4(AQP4); members of the complement cascade; ATPase, H+ transporting,lysosomal V1 subunit A (ATP6V1A); gap junction protein α-1 (GJA1);formyl peptide receptor 1 (FPR1); formyl peptide receptor-like 1(FPRL1); interleukin 8 (IL8); nuclear factor kappa-B, subunit 1 (NFKB1);nuclear factor kappa-B, subunit 2 (NFKB2); presenilin 1 (PSEN1); tumornecrosis factor-alpha-converting enzyme (TACE); transforming growthfactor β2 (TGFB2); transient receptor potential cation channel,subfamily V, member 1 (TRPV1); chloride channel 3 (CLCN3); gap junctionprotein α5 (GJA5); tumor necrosis factor receptor 1 (TNFR1); andchitinase 3-like 2 (CHI3L2).

Examples of mRNA target genes associated with ocular inflammationinclude tumor necrosis factor receptor superfamily, member 1A(TNFRSF1A); phosphodiesterase 4D, cAMP-specific (PDE4D); histaminereceptor H1 (HRH1); spleen tyrosine kinase (SYK); interleukin 1β (IL1B);nuclear factor kappa-B, subunit 1 (NFKB1); nuclear factor kappa-B,subunit 2 (NFKB2); and tumor necrosis factor-alpha-converting enzyme(TACE).

Such target genes are described, for example, in U.S. patentapplications having Publication Nos. 20060166919, 20060172961,20060172963, 20060172965, 20060223773, 20070149473, and 20070155690, thedisclosures of which are incorporated by reference in their entirety.

In certain embodiments, the invention provides a pharmaceuticalcomposition comprising an interfering RNA delivery system of theinvention. In certain embodiments, the composition is in apharmaceutically acceptable carrier in a therapeutically effectiveamount.

Pharmaceutical compositions are formulations that comprise interferingRNAs, or salts thereof, of the invention up to 99% by weight mixed witha physiologically acceptable carrier medium, including those describedinfra, and such as water, buffer, saline, glycine, hyaluronic acid,mannitol, and the like.

Compositions of the present invention are administered as solutions,suspensions, or emulsions. The following are examples of pharmaceuticalcomposition formulations that may be used in the methods of theinvention.

Amount in weight % Interfering RNA up to 99; 0.1-99; 0.1-50; 0.5-10.0Hydroxypropylmethylcellulose 0.5 Sodium chloride 0.8 BenzalkoniumChloride 0.01 EDTA 0.01 NaOH/HCl qs pH 7.4 Purified water (RNase-free)qs 100 mL

Amount in weight % Interfering RNA up to 99; 0.1-99; 0.1-50; 0.5-10.0Phosphate Buffered Saline 1.0 Benzalkonium Chloride 0.01 Polysorbate 800.5 Purified water (RNase-free) q.s. to 100%

Amount in weight % Interfering RNA up to 99; 0.1-99; 0.1-50; 0.5-10.0Monobasic sodium phosphate 0.05 Dibasic sodium phosphate 0.15(anhydrous) Sodium chloride 0.75 Disodium EDTA 0.05 Cremophor EL 0.1Benzalkonium chloride 0.01 HCl and/or NaOH pH 7.3-7.4 Purified water(RNase-free) q.s. to 100%

Amount in weight % Interfering RNA up to 99; 0.1-99; 0.1-50; 0.5-10.0Phosphate Buffered Saline 1.0 Hydroxypropyl-β-cyclodextrin 4.0 Purifiedwater (RNase-free) q.s. to 100%

As used herein, the term “therapeutically effective amount” refers tothe amount of interfering RNA or a pharmaceutical composition comprisingan interfering RNA determined to produce a therapeutic response in amammal Such therapeutically effective amounts are readily ascertained byone of ordinary skill in the art and using methods as described herein.

Generally, a therapeutically effective amount of the interfering RNAsused in a composition of the invention results in an extracellularconcentration at the surface of the target cell of from 100 pM to 1 μM,or from 1 nM to 100 nM, or from 5 nM to about 50 nM, or to about 25 nM.The dose required to achieve this local concentration will varydepending on a number of factors including the delivery method, the siteof delivery, the number of cell layers between the delivery site and thetarget cell or tissue, whether delivery is local or systemic, etc. Theconcentration at the delivery site may be considerably higher than it isat the surface of the target cell or tissue. Topical compositions can bedelivered to the surface of the target organ one to four times per day,or on an extended delivery schedule such as daily, weekly, bi-weekly,monthly, or longer, according to the routine discretion of a skilledclinician. The pH of the formulation is about pH 4.0 to about pH 9.0, orabout pH 4.5 to about pH 7.4.

A therapeutically effective amount of a formulation may depend onfactors such as the age, race, and sex of the subject, the severity ofthe disorder, the rate of target gene transcript/protein turnover, theinterfering RNA potency, and the interfering RNA stability, for example.In one embodiment, the interfering RNA is delivered topically to atarget organ and reaches the target mRNA-containing tissue at atherapeutic dose thereby ameliorating target gene-associated diseaseprocess.

Therapeutic treatment of patients with interfering RNAs directed againsttarget mRNAs is expected to be beneficial over small molecule treatmentsby increasing the duration of action, thereby allowing less frequentdosing and greater patient compliance, and by increasing targetspecificity, thereby reducing side effects.

A “pharmaceutically acceptable carrier” as used herein refers to thosecarriers that cause at most, little to no irritation, provide suitablepreservation if needed, and deliver one or more interfering RNAs of thepresent invention in a homogenous dosage.

The delivery systems and compositions of the present invention may bedelivered in solution, in suspension, or in bioerodible ornon-bioerodible delivery devices.

The delivery systems and compositions of the present invention may bedelivered via absorption, adsorption, aerosol, buccal, dermal, inhaling,intracentricular, intracranial, intradermal, intramuscular, intranasal,intraocular, intrapulmonary, intravenous, intraperitoneal, intrasternal,intrathecal, intraventricular, nasal, ocular, oral, otic, parenteral,patch, rectal, systemic, subcutaneous, sublingual, topical, ortransdermal, or vaginal administration, for example.

Interfering RNA delivery systems may be delivered directly to the eye byocular tissue injection such as periocular, conjunctival, subtenon,intracameral, intravitreal, intraocular, anterior or posteriorjuxtascleral, subretinal, subconjunctival, retrobulbar, orintracanalicular injections; by direct application to the eye using acatheter or other placement device such as a retinal pellet, intraocularinsert, suppository or an implant comprising a porous, non-porous, orgelatinous material; by topical ocular drops or ointments; or by a slowrelease device in the cul-de-sac or implanted adjacent to the sclera(transscleral) or in the sclera (intrascleral) or within the eye.Intracameral injection may be through the cornea into the anteriorchamber to allow the agent to reach the trabecular meshwork.Intracanalicular injection may be into the venous collector channelsdraining Schlemm's canal or into Schlemm's canal.

For ocular administration, the compositions of the invention can bedelivered by intravitreal injection every 2-6 weeks, for example, or viatopical ocular, anterior or posterior juxtascleral depot,subconjunctival, periocular, retrobulbar, subtenon, intracameral,intraocular, subretinal, or suprachoroidal administration.

For pharmaceutical delivery, compositions of the present invention maybe combined with pharmaceutically acceptable preservatives, co-solvents,surfactants, viscosity enhancers, penetration enhancers, buffers, sodiumchloride, or water to form an aqueous, sterile suspension or solution.Solution formulations may be prepared by dissolving the conjugate in aphysiologically acceptable isotonic aqueous buffer. Further, thesolution may include an acceptable surfactant to assist in dissolvingthe interfering RNA. Viscosity building agents, such as hydroxymethylcellulose, hydroxyethyl cellulose, methylcellulose,polyvinylpyrrolidone, or the like may be added to the compositions ofthe present invention to improve the retention of the compound.

In order to prepare a sterile ointment formulation, the composition iscombined with a preservative in an appropriate vehicle, such as mineraloil, liquid lanolin, or white petrolatum. Sterile gel formulations maybe prepared by suspending the composition of the invention in ahydrophilic base prepared from the combination of, for example,CARBOPOL®-940 (BF Goodrich, Charlotte, N.C.), or the like, according tomethods known in the art. VISCOAT® (Alcon Laboratories, Inc., FortWorth, Tex.) may be used for intraocular injection, for example. Othercompositions of the present invention may contain penetration enhancingagents such as cremephor and TWEEN® 80 (polyoxyethylene sorbitanmonolaureate, Sigma Aldrich, St. Louis, Mo.).

In certain embodiments, the invention also provides a kit that includesreagents for attenuating the expression of an mRNA as cited herein in acell. The kit contains an interfering RNA molecule conjugated to aCPP-Arg or a CPP-HA2-Arg peptide and/or the necessary components forproduction of an interfering RNA molecule conjugated to a CPP-Arg or aCPP-HA2-Arg peptide (e.g., an interfering RNA molecule as well as thepeptide and necessary materials for linking). The kit may also containpositive and negative control siRNAs or shRNA expression vectors (e.g.,a non-targeting control siRNA or an siRNA that targets an unrelatedmRNA). The kit also may contain reagents for assessing knockdown of theintended target gene (e.g., primers and probes for quantitative PCR todetect the target mRNA and/or antibodies against the correspondingprotein for western blots). Alternatively, the kit may comprise an siRNAsequence or an shRNA sequence and the instructions and materialsnecessary to generate the siRNA by in vitro transcription or toconstruct an shRNA expression vector.

A pharmaceutical combination in kit form is further provided thatincludes, in packaged combination, a carrier means adapted to receive acontainer means in close confinement therewith and a first containermeans including an interfering RNA composition and a peptide. Such kitscan further include, if desired, one or more of various conventionalpharmaceutical kit components, such as, for example, containers with oneor more pharmaceutically acceptable carriers, additional containers,etc., as will be readily apparent to those skilled in the art. Printedinstructions, either as inserts or as labels, indicating quantities ofthe components to be administered, guidelines for administration, and/orguidelines for mixing the components, can also be included in the kit.

The references cited herein, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated by reference.

Those of skill in the art, in light of the present disclosure, willappreciate that obvious modifications of the embodiments disclosedherein can be made without departing from the spirit and scope of theinvention. All of the embodiments disclosed herein can be made andexecuted without undue experimentation in light of the presentdisclosure. The full scope of the invention is set out in the disclosureand equivalent embodiments thereof. The specification should not beconstrued to unduly narrow the full scope of protection to is which thepresent invention is entitled.

EXAMPLES

The following example, including the experiments conducted and resultsachieved is provided for illustrative purposes only and are not to beconstrued as limiting the invention.

Example 1 Delivery of Dy547-siRNA to GTM-3 Cells Using 9× Arg-LinkedPen7 Peptide

The ability of CPPs to facilitate cellular uptake of siRNA molecules wasexamined using a retro-inverso 7-amino acid fragment of penetratin(Pen7), conjugated to a Dy547-labeled siRNA (siGLO Cyclophilin B ControlsiRNA; Dharmacon, Lafayette, Colo.) via 9× Arg, for delivery to theglaucomatous trabecular meshwork cell line, GTM-3.

GTM-3 cells (Pang, I. H., et al., 1994 Curr Eye Res. 13:51-63) weretransfected with Dy547-siRNA complexed with Pen7-9× Arg or with 9× Arg(negative control). The 9× Arg and Pen7-9× Arg peptides were purchasedfrom Sigma (St. Louis, Mo.).

The sequence for Pen7-9× Arg was:

(SEQ ID NO: 12)K^(†)K^(†)W^(†)K^(†)M^(†)R^(†)R^(†)GA^(†)GR^(†)R^(†)R^(†)R^(†)R^(†)R^(†)R^(†)R^(†)R^(†).

The sequence for 9× Arg was:

(SEQ ID NO: 1) 9xArg: R^(†)R^(†)R^(†)R^(†)R^(†)R^(†)R^(†)R^(†)R^(†).

A superscripted dagger (^(†)) to the right of the one-letter code symboldenotes the use of a D-amino acid isomer as opposed to the standardL-amino acid isomer.

The Dy547-siRNA was resuspended in 1× siRNA buffer, an aqueous solutionof 20 mM KCl, 6 mM HEPES (pH 7.5), 0.2 mM MgCl₂. siRNA-peptide complexeswere prepared at a 1:10 molar ratio of siRNA to peptide, and incubatedfor 30 minutes at room temperature. The siRNA-peptide complexes wereapplied to GTM-3 cells in serum-free medium at a final siRNAconcentration of 400 nM. After 4 hours, the medium was replaced withDMEM supplemented with 10% FBS. After 24 hours, the cells wereharvested, and uptake of Dy547-siRNA was measured in a LSRII flowcytometry (BD Biosciences, Franklin Lakes, N.J.).

As shown in FIG. 1, GTM-3 cells were labeled only slightly by theDy547-siRNA in the absence of a peptide (FIG. 1B) relative tonon-treated cells (FIG. 1A). Addition of the 9× Arg peptide to theDy547-siRNA did not enhance uptake (FIG. 1C) relative to the Dy547-siRNAalone (FIG. 1B). In contrast, Pen7-9× Arg peptide enhanced uptake of theDy547-siRNA significantly, causing an increased fluorescence signal inapproximately 88% of the cells (FIG. 1D).

These results demonstrated that linkage of siRNAs to CPP peptidesfacilitated siRNA delivery to cultured cells.

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims.

What is claimed is:
 1. An interfering RNA delivery system comprising aninterfering RNA molecule linked to a cell penetrating peptide (CPP)-Argpeptide.
 2. The delivery system of claim 1, wherein the CPP-Arg peptidefurther comprises an HA2 peptide.
 3. The delivery system of claim 1,wherein the interfering RNA molecule is linked to the CPP-Arg peptidevia a covalent bond.
 4. The delivery system of claim 1, wherein theinterfering RNA molecule is a siRNA, miRNA, or shRNA.
 5. Apharmaceutical composition comprising the delivery system of claim 1 anda pharmaceutically acceptable carrier.
 6. The delivery system of claim1, wherein the CPP is a TAT peptide or cell penetrating fragmentthereof, or the protein transduction domain of Penetratin (pAntp),Transportan, MPG, MPGdeltaNLS, or pHLIP, or a cell penetrating fragmentthereof.
 7. The delivery system of claim 1, wherein the CPP has an aminoacid sequence in the forward or reverse direction.
 8. The deliverysystem of claim 7, wherein the CPP comprises at least one D-amino acid.9. The delivery system of claim 1, wherein the CPP-Arg peptide comprisesthe sequence as set forth in SEQ ID NO:
 12. 10. A method of treating orpreventing an ocular disorder in a patient, comprising administering tothe patient the interfering RNA delivery system of claim 1, wherein theinterfering RNA molecule can attenuate expression of a gene associatedwith the ocular disorder.
 11. The method of claim 10, wherein the oculardisorder is associated with ocular angiogenesis, dry eye, ocularinflammatory conditions, ocular hypertension, or glaucoma.
 12. Themethod of claim 10, wherein the system is administered by intraocularinjection, ocular topical application, subconjunctival injection,intravitreal injection, anterior or posterior juxtascleral injection,intravenous injection, oral administration, intramuscular injection,intraperitoneal injection, transdermal application, intranasalapplication, or transmucosal application.
 13. A method of delivering aninterfering RNA molecule into a cell, comprising: (a) attaching aninterfering RNA molecule to a CPP-Arg peptide, thereby forming aninterfering RNA delivery system; and (b) administering the interferingRNA delivery system to the cell under conditions suitable for the systemto enter the cell.
 14. The method of claim 13, wherein release of thesystem from the endosome inside the cell is enhanced in the presence ofan HA2 peptide.
 15. The method of claim 13, wherein the HA2 peptide isintroduced into the cell by administration of a CPP-HA2 peptide.
 16. Themethod of claim 13, wherein the CPP-Arg peptide further comprises theHA2 peptide.
 17. The method of claim 13, wherein the interfering RNAmolecule is linked to the CPP-Arg peptide via a covalent bond.
 18. Themethod of claim 13, wherein the interfering RNA molecule is a siRNA,miRNA, or shRNA.
 19. The method of claim 13, wherein the CPP is a TATpeptide or cell penetrating fragment thereof, or the proteintransduction domain of Penetratin (pAntp), Transportan, MPG,MPGdeltaNLS, or pHLIP, or a cell penetrating fragment thereof.
 20. Themethod of claim 13, wherein the CPP-Arg peptide comprises the sequenceas set forth in SEQ ID NO:
 12. 21. The method of claim 13, wherein theCPP has an amino acid sequence in the forward or reverse direction. 22.The method of claim 21, wherein the CPP comprises at least one D-aminoacid.